Does RNA - PCR ??
sehuang at aol.com
Sun Jul 23 19:08:02 EST 1995
Several people in reponses to the problem of PCR on RNA mentioned a RT
activity of thermostable DNA pol. I think it is rather contamination with
DNA that gives rise to the products. Even after rigorous DNAse treatment
and digestion with 4-cutters restriction enzymes one might be able to
amplify DNA in PCR. I had an artificial gene with an artificial
mini-intron that allowed me to clearly distinguish between DNA (slightly
longer amplicon due to intron) and RNA, and it was the DNA that is
amplified. The band occured in the same strength in the No-RT control. (Or
was it unspliced RNA ???)
Nevertheless I am highly interested in knowing more about the RT activity
of Taq pol. I still cannot understand how the RNA:DNA hybrid generated
after the first round of PCR by Taq pol should denature at 94 degree C (ie
denaturation step in PCR ). RNA:DNA hybrids are more stable than DNA:DNA
duplex and usually do not separate at 90 dgree as I remember. Does the
Taq also has an RNAse H activity, like conventional reverse Transcriptases
Has anyone literarture about that ?
Thank you for any suggestions, comments.
More information about the Methods